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Vol. 171, No. 1
JOURNAL OF BACTERIOLOGY, Jan. 1989, p. 569-572
0021-9193/89/010569-04$02.00/0
Copyright © 1989, American Society for Microbiology
Roles of Flagella, Lipopolysaccharide, and a Ca2 -Dependent Cell
Surface Protein in Attachment of Rhizobium leguminosarum
Biovar viciae to Pea Root Hair Tips
GERRIT SMIT,* JAN W. KIJNE, AND BEN J. J. LUGTENBERG
Department of Plant Molecular Biology, Leiden University, 2311 VJ Leiden, The Netherlands
Received 17 March 1988/Accepted 27 September 1988
The relationship between Ca2+-dependent cell surface components of Rhizobium leguminosarum biovar
viciae, motility, and ability to attach to pea root hair tips was investigated. In contrast to flagella and
lipopolysaccharide, a small protein located on the cell surface was identified as the Ca2+-dependent adhesin.
flagellumless cells that also lacked the major 32-kDa flagellin
band in the crude flagellum prfparation (strains RBL1484
through RBL1495); class 2 consisted of mutants that still
possessed flagella but lacked an 18-kDa band present in the
crude flagellum preparation (strains RBL1496 through
RBL1507); and class 3 consisted of mutants possessing
flagella and with a gel electrophoresis pattern of the crude
flagellum preparations similar to that of the wild-type strain
(RBL1508 through RBL1516) (Fig. 1). Mutants from classes
1 and 3 were indistinguishable from the wild-type strain with
respect to attachment and nodulation ability on pea and
common vetch, which indicated that motility and exposure
of flagella are not essential for nodulation of R. leguminosa-
Attachment of rhizobia to developing root hairs is one of
the first steps of the nitrogen-fixing root nodule symbiosis
between rhizobia and the leguminous host plants. Recently,
we reported that both cellulose fibrils and a Ca2+-dependent
adhesin of Rhizobium leguminosarum bv. viciae cells are
involved in the two-step process of attachment of rhizobia to
pea root hair tips (13). In the study reported here, the
influence of Ca2" limitation on motility and surface components of R. leguminosarum cells is described in relation to
the ability of the cells to attach to pea root hair tips, and the
Ca2"-dependent adhesin is identified as a small cell surface
protein.
Ca2+ is essential for motility of R. leguminosarum. Attachment ability (13) and motility of R. leguminosarum 248,
harboring Sym plasmid pRLlJI (9), were found to decrease
strongly under low-Ca2" conditions. No motility was observed when the Ca2+ concentration in TY medium (12) was
below 1.4 mM, whereas the growth rate was not affected. An
electron microscopic study of rhizobia grown under Ca2`
limitation (13) showed that flagella were not present on the
cell surface.
Purified flagella are not involved in attachment of R. keguminosarum. To determine the possible role of flagella as well
as motility in attachment of rhizobia, the adhesin activity of
purified flagella and the attachment ability of nonmotile
mutants were determined. Flagella from R. leguminosarum
248, purified according to the method of Carsiotis et al. (5),
appeared to be 12 to 13 nm in diameter and up to 4 ,um long
as judged by electron microscopy. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (11) of purified flagella showed a dominant 32-kilodalton (kDa) protein
(Fig. 1, lane I). Crude flagellum preparations from R. leguminosarum 248 grown at various Ca2" concentrations, obtained as described above but without the density centrifugation step, showed that under low-Ca2" conditions the 32
kDa band was missing, whereas the densities of several
other bands had increased (data not shown). These data
demonstrate that the 32-kDa protein represents the flagellar
subunit of R. leguminosarum.
Thirty-three nonmotile TnS mutants of R. leguminosarum
248 were isolated (6, 13) and examined for the presence of
flagella by electron microscopy, and crude flagellum preparations isolated from these mnutants were investigated by
using SDS-PAGE. The nonmotile mutants could be divided
into three classes. Class 1 contained mutants consisting of
*
rum.
An adhesin was experimentally defined as a surface component of rhizobia able to inhibit attachment of rhizobial
cells to pea root hairs when supplied before or during an
attachment assay. Attachment of R. leguminosarum was
affected neither by incubation of the roots with purified
flagella before incubation with the bacteria (Table 1) nor by
addition of flagella during the attachment assay (data not
shown). Taken together, these results demonstrate that
flagella are not involved in attachment of R. leguminosarum
and that reduced attachment ability as a result of Ca2"
limitation is not due to loss of flagella or motility.
Attachment of nonmotile mutants affected in lipopolysaccharide composition. Twelve nonmotile mutants were found
to lack an 18-kDa band as judged by SDS-PAGE of crude
flagellum preparations derived from these strains (Fig. 1,
lanes D through F). These nonmotile mutants appeared to be
LPS mutants. LPS was isolated, according to the method of
Westphal and Jann (15), from the wild-type strain and from
a number of class 2 mutants. Analysis of LPS from R.
leguminosarum 248 by SDS-PAGE revealed two bands of
differing molecular masses running at positions of proteins
with apparent molecular masses of 18 and 12 kDa, respectively (Fig. 2, lane A). Similar results were described by
Carlson et al. (3, 4) for LPS of R. leguminosarum biovars
trifolii and phaseoli; in their studies, the lower-molecularweight band appeared to represent the lipid A and core part
of the LPS and the higher-molecular-weight form appeared
to represent the complete LPS, consisting of lipid A, core,
and 0-antigenic polysaccharide. LPS isolated from the class
2 mutants RBL1496, RBL1497, and RBL1500 appeared to
lack the high-molecular-weight form of the LPS (Fig. 2, lanes
B and C) and therefore most likely the 0-antigenic polysaccharide part of the LPS and perhaps part of the core. Mutant
Corresponding author.
569
570
NOTES
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TABLE 1. Influence of various Ca2"-dependent cell surface
components from R. leguminosarum 248 on attachment of
R. leguminosarum 248 cells to pea root hair tipsa
IPW
-.
-
Deviation from
standard assay
_
IV IF
,W_
mo
OD
4
28
25
28
12
13
16
50
54
50
7.0mM Ca2+
0,35 mM Ca2+
47
13
26
19
19
9
8
59
CSP, 7.0 mM Ca2+
Heat treatedd
Protease treatede
Proteasee
9
3
15
30
31
16
6
11
11
55
55
58
CSP 7.0 mM Ca2+f
>30 kDa
<30 kDa
>5 kDa
<5 kDa
17
50
39
12
16
22
33
19
12
11
12
9
55
17
16
60
CsPc
_
% Attachment in class:b
3
2
10
8
6
None
Flagella
LPS
ON
1
FIG. 1. SDS-PAGE of crude flagellum preparations of R. leguminosarum 248 and some nonmotile mutants. Lanes: A, R. leguminosarum 248; B, RBL1484 (class 1); C, RBL1485 (class 1); D,
RBL1496 (class 2); E, RBL1497 (class 2); F, RBL1S00 (class 2); G,
RBL1508 (class 3); H, RBL1509 (class 3); I, purified flagella of R.
leguminosarum 248. Positions of molecular mass markers are indicated at the left (size given in kilodaltons). Symbols: D, flagellin;
,, higher-molecular-weight form of the LPS. Note that the
staining procedure used (2) stains both proteins and LPSs.
a Bacteria was harvested at an A620 of 0.70, suspended, and added to the
pea roots in a final concentration of 1.5 x 108 to 2.0 x 108 cells per ml (12).
Roots were incubated with flagella (100 FgIml), LPS (250 ,ug/ml), cell surface
preparation (CSP), proteinase K (200 ,ug/ml), or potassium phosphate buffer
for 60 min, washed, and incubated with the bacteria.
I Class 1, No attached bacteria; class 2, few attached bacteria; class 3, the
apical portion of the root hair covered with bacteria; class 4, many attached
bacteria forming a caplike aggregate on top of the root hair.
Cell surface preparations (200 ,ul) derived from 10 ml of R. leguminosarum
248 culture, grown at Ca2+ concentrations of 7.0 and 0.35 mM, were added to
the roots.
d Cell surface preparation derived from rhizobia grown under normal Ca2+
conditions was incubated at 100°C for 5 min before incubation with the roots.
eCell surface preparation was incubated with proteinase K (1 mg/ml) at
37°C for 60 min before incubation with the roots. As a control, roots were
incubated for 60 min at room temperature with proteinase K before incubation
with bacteria.
f Cell surface preparations were separated into two fractions by ultrafiltration, using a 30- and a 5-kDa membrane. Equal amounts corresponding to a
cell surface preparation derived from 10 ml of culture were used in the
RBL1500 was an exception in that it was found to yield an
additional band (Fig. 2, lane D). This result might be
attributable to a reduced length of the 0-antigenic polysaccharide part of the LPS or to a lack of putative side chains in
the 0-antigenic repeating unit. These results indicate that the
LPS of R. leguminosarum is involved in motility of the
bacteria, as has been found for LPS mutants of other
gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium (1, 7).
Since LPS has repeatedly been proposed to be involved in
attachment of members of the family Rhizobiaceae to host
plant cells (8, 10, 16) and since the 18-kDa band in crude
flagellum preparations was found to strongly increase under
Ca2'-limiting conditions, we studied the possibility that LPS
is involved in attachment to pea- root hairs. LPS isolated
from R. leguminosarum 248 and from strains RBL1496,
RBL1497, and RBL1500, added to the roots in concentrations of up to 250 jig/ml before the attachment assay, did not
inhibit attachment of R. leguminosarum 248 (Table 1). In
attachment assays in which the LPS was added during the
attachment assay, the size of the caps (class 4 attachment;
12) was even increased (data not shown). With one exception, attachment of LPS mutants of R. leguminosarum was
similar to that of the wild-type strain 248. However, since
LPS mutants were found to adhere optimally to pea root
hairs at earlier phases during growth in batch culture than
did the wild-type strain, the LPS might be involved indirectly in attachment, e.g., in masking of adhesins on the cell
surface of the bacteria. Comparable results were found for
0-antigen-less LPS mutants of uropathogenic E. coli (14).
The LPS mutants nodulated pea and common vetch, although nodulation on the latter host plant was delayed for 3
to 7 days. Taken together, these results demonstrate that
LPS is not directly involved in the attachment process.
One LPS mutant, strain RBL1500, showed a reduced
ability to attach to pea root hair tips. This strain was found
to be affected in the second step of the attachment process,
and since cellulose fibril isolation (13) revealed that this
mutant does not produce cellulose fibrils, it is very likely that
this pleiotropic effect causes the altered phenotype with
respect to attachment.
The Ca2"-dependent adhesin of Rhizobwum appears to be a
soluble surface protein. The supernatant, and not the flagellum-containing pellet, obtained after the ultracentrifugation
step in flagellum purification appeared to possess attachment-inhibiting activity (Table 1), which indicated that this
fraction contained an adhesin which was detached from the
bacteria together with the flagella. This fraction is called the
cell surface preparation. Adhesin activity was found both
when the cell surface preparation was incubated with the pea
roots before the attachment assay as well as during the
7
18K
A B C
D E
F
G
4
HI
experiments.
NOTES
VOL. 171, 1989
571
of R. leguminosarum to pea root hair tips (Table 1). Treatment of the cell surface preparation with proteolytic enzymes for 60 min at 370C also resulted in loss of attachmentinhibiting activity of the adhesin. A control incubation of the
roots with protease was necessary, since protease could not
easily be removed from the cell surface preparation after the
treatment. This control incubation did not affect attachment
(Table 1). Ultrafiltration of the cell surface preparation
yielded a molecular mass for the adhesin of between 5 and 30
kDa (Table 1). Taken together, these results indicate that the
adhesin is a Ca2"-dependent, cell surface-located, watersoluble, heat-labile small protein.
Future research will focus on purification and characterization of the Ca2"-dependent adhesin and on isolation of
mutants lacking this adhesin.
-~~~~~
This investigation was supported by the Foundation for Fundamental Biological Research, which is subsidized by the Netherlands
Organization for Advancement of Pure Research.
We thank Trudy Logman and Chantal Rust for their contributions
to this work.
1.
2.
3.
A
B
C
D
FIG. 2. SDS-PAGE of isolated LPS of R. leguminosarum 248
(lane A), RBL1496 (lane B), RBL1497 (lane C), and RBL1500 (lane
D). Equal amounts (2.5 ,ug) of LPS were applied in all slots.
Symbols: >, higher-molecular-weight form; *, lower-molecularweight form.
4.
5.
attachment assay, although in the former case attachmentinhibiting activity was higher, a result most likely due to a
lack of competition between the adhesin and the bacteria.
The attachment-inhibiting factor resulted in a high percentage of root hairs without attached bacteria (Table 1), which
indicated that this factor is involved in the first step of the
attachment process (see also reference 13). Cell surface
preparations isolated from representatives of the three nonmotile mutant classes, including strain RBL1500, were all
found to possess attachment-inhibiting activity, which indicated that none of the nonmotile mutants was affected in the
synthesis of this adhesin (data not shown).
To determine whether the adhesin present in the cell
surface preparation is Ca2" dependent, a cell surface preparation was isolated from R. leguminosarum 248 grown
under low-Ca2+ conditions. This fraction did not possess
any attachment-inhibiting activity (Table 1), which makes it
very likely that the adhesin present in the cell surface
preparation was the Ca2+-dependent adhesin which mediates the first step in Rhizobium attachment.
Partial characterization of the adhesin revealed that it
must be a soluble surface component, since no activity was
found in the pellet fraction even after prolonged ultracentrifugation for up to 4 h at 100,000 x g. Treatment of a cell
surface preparation by heat for 5 min at 100°C completely
abolished the ability of the preparation to inhibit attachment
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